U.S. patent number 5,675,517 [Application Number 08/428,720] was granted by the patent office on 1997-10-07 for fluorescence spectral overlap compensation for high speed flow cytometry systems.
This patent grant is currently assigned to Systemix. Invention is credited to Willem Stokdijk.
United States Patent |
5,675,517 |
Stokdijk |
October 7, 1997 |
**Please see images for:
( Certificate of Correction ) ** |
Fluorescence spectral overlap compensation for high speed flow
cytometry systems
Abstract
A fluorescence spectral overlap electronic compensation circuit
for high speed flow cytometry systems is provided. In high-speed
flow cytometry systems, such as systems with a pulse rise time up
to 2 .mu.s, baseline restoration circuits may not adequately
eliminate the DC offset of input signals, in which case, DC offset
will result in errors after overlap compensation. In addition,
analog spectral overlap compensation operations may result in
signal distortions that are unacceptable when using a log amp whose
output signal depends on the absolute value of its input signal.
The disclosed fluorescence spectral overlap compensation circuit
includes an adjustable DC offset compensation circuit to more
accurately reduce the DC offset components of signals, and a
half-wave rectifier for eliminating the signal distortions caused
by spectral overlap compensation operations.
Inventors: |
Stokdijk; Willem (Livermore,
CA) |
Assignee: |
Systemix (Palo Alto,
CA)
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Family
ID: |
23700111 |
Appl.
No.: |
08/428,720 |
Filed: |
April 25, 1995 |
Current U.S.
Class: |
702/85;
356/338 |
Current CPC
Class: |
G01N
15/14 (20130101) |
Current International
Class: |
G01N
15/14 (20060101); G01C 025/00 () |
Field of
Search: |
;364/571.01,571.02,555,550 ;327/307,309
;356/349,338,340,364,441,442,39,341,342,343,335,336,432-437
;250/564,565,573 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0585754 |
|
Mar 1994 |
|
EP |
|
0641573 |
|
Mar 1995 |
|
EP |
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Other References
Hiebert, R. D. "Electronics and Signal Processing", Flow Cytometry
and Sorting, Second Edition, pp. 127-144, 1990 Wiley-Liss Inc.
.
Loken, Michael R., David R. Parks and Leonard A. Herzenberg
"Two-Color Immunofluorescence using a Fluorescence-Activated Cell
Sorter", The Journal of Histochemistry and Cytochemistry, vol. 25,
No. 7, 1977, pp. 899-907. .
Bagwell, C. Bruce and Earl G. Adams, "Fluorescence Spectral Overlap
Compensation for Any Number of Flow Cytometry Parameters", Annals
New York Academy of Sciences, pp. 167-184 date unknown..
|
Primary Examiner: Trammell; James P.
Attorney, Agent or Firm: Blakely, Sokoloff, Taylor &
Zafman
Claims
What is claimed is:
1. A preamplifier for use in a flow cytometry system, the
preamplifier comprising:
a baseline restoration circuit coupled to receive a voltage-encoded
signal, the baseline restoration circuit generating an estimated DC
component responsive to the voltage-encoded signal; and
an offset compensation circuit coupled to the baseline restoration
circuit, the offset compensation circuit generating an offset
signal indicating a voltage offset; and
wherein the preamplifier is configured to attenuate the
voltage-encoded signal based on the voltage offset and the
estimated DC component to produce a DC compensated signal.
2. The preamplifier of claim 1 wherein:
the baseline restoration circuit generates a DC component signal
having a voltage level equal to the offset signal summed with the
estimated DC component; and
the DC compensated signal is generated by attenuating the
voltage-encoded signal based on the DC component signal.
3. The preamplifier of claim 1 wherein said offset compensation
circuit includes an offset compensation level adjustment mechanism
that allows a user to set the voltage offset indicated by said
offset signal.
4. The preamplifier of claim 3 wherein said offset compensation
level adjustment mechanism includes a potentiometer.
5. A data acquisition system comprising a preamplifier as recited
in claim 1, the data acquisition system further comprising:
a detector disposed to detect particles having a first tag type,
the detector generating an analog current which varies responsive
to detecting particles having the first tag type;
a current-to-voltage converter coupled to the detector, the
current-to-voltage converter receiving the analog current from said
detector and generating the voltage-encoded signal responsive to
the analog current;
an amplifier coupled to the preamplifier, the amplifier amplifying
the DC compensated signal and generating an amplified signal;
an analog-to-digital converter coupled to the amplifier, the
analog-to-digital converter generating digital data responsive to
the amplified signal; and
a computer coupled to the analog-to-digital converter, the computer
categorizing the particles responsive to the digital data.
6. The data acquisition system of claim 5 wherein said particles
are cells.
7. A data acquisition system comprising:
a detector disposed to detect particles having a first tag type,
the detector generating an analog current which varies responsive
to detecting particles having the first tag type;
a current-to-voltage converter coupled to the detector, the
current-to-voltage converter receiving the analog current from said
detector and generating the voltage-encoded signal responsive to
the analog current;
a preamplifier comprising:
a baseline restoration circuit coupled to receive a voltage-encoded
signal, the baseline restoration circuit generating an estimated DC
component responsive to the voltage-encoded signal; and
an offset compensation circuit coupled to the baseline restoration
circuit, the offset compensation circuit generating an offset
signal indicating a voltage offset; and
wherein the preamplifier is configured to attenuate the
voltage-encoded signal based on the voltage offset and the
estimated DC component to produce a DC compensated signal;
an amplifier coupled to the preamplifier, the amplifier amplifying
the DC compensated signal and generating an amplified signal;
an analog-to-digital converter coupled to the amplifier, the
analog-to-digital converter generating digital data responsive to
the amplified signal;
a computer coupled to the analog-to-digital converter, the computer
categorizing the particles responsive to the digital data;
a second detector disposed to detect particles having a second tag
type, the second detector generating a second analog current which
varies responsive to detecting particles having the second tag
type;
a reduced signal transmission circuit coupled to the second
detector, the reduced signal transmission circuit receiving the
second analog current and generating a reduced level signal, the
reduced level signal being the second analog current reduced by a
percentage;
an overlap compensation circuit coupled between the preamplifier
and the amplifier, the overlap compensation circuit being coupled
to the reduced signal transmission circuit, the overlap
compensation circuit subtracting the reduced level signal from the
DC compensated signal to produce an overlap compensated signal;
and
a half-wave rectifier coupled between the overlap compensation
circuit and the amplifier, the half-wave rectifier receiving the
overlap compensated signal and generating a rectified signal, the
amplifier amplifying the rectified signal to produce the amplified
signal.
8. A spectral overlap compensation system for use in a flow
cytometry system, the spectral overlap compensation system
comprising:
an overlap compensation circuit coupled to a first detector, said
overlap compensation circuit receiving a first signal from said
first detector, the overlap compensation circuit subtracting a
second signal from the first signal to produce an overlap
compensated signal, wherein the second signal is based on signals
produced by one or more second detectors;
a half-wave rectifier coupled to the overlap compensation circuit,
the half-wave rectifier receiving the overlap compensated signal
and generating a rectified signal; and
an amplifier coupled to the half-wave rectifier, the amplifier
amplifying the rectified signal and generating an amplified
signal.
9. The spectral overlap compensation circuit of claim 8 wherein
said amplifier is a logarithmic amplifier configured to generate
said amplified signal based on the absolute value of said overlap
compensated signal.
10. The spectral overlap compensation system of claim 8 wherein
said overlap compensation circuit is coupled to said first detector
through a preamplifier circuit, wherein said preamplifier circuit
includes:
a current-to-voltage converter coupled to the first detector, the
current-to-voltage converter receiving an analog current from said
first detector and generating a voltage-encoded signal responsive
to the analog current;
a baseline restoration circuit coupled to the current-to-voltage
converter, the baseline restoration circuit generating an estimated
DC component responsive to the voltage-encoded signal;
an offset compensation circuit coupled to the baseline restoration
circuit, the offset compensation circuit generating an offset
signal indicating a voltage offset; and
the preamplifier circuit being configured to attenuate the
voltage-encoded signal based on the voltage offset and the
estimated DC component to produce said first signal.
11. A data acquisition system that includes a spectral overlap
compensation system as recited in claim 8, the data acquisition
system further comprising:
said first detector disposed to detect particles having a first tag
type, the first detector generating a first analog current which
varies responsive to detecting particles having the first tag type,
wherein said first signal is based on said first analog
current;
a second detector disposed to detect particles having a second tag
type, the second detector generating a second analog current which
varies responsive to detecting particles having the second tag
type;
a reduced signal transmission circuit coupled to the second
detector, the reduced signal transmission circuit receiving the
second analog current and generating said second signal, the second
signal representing the second analog current reduced by a
percentage;
an analog-to-digital converter coupled to the amplifier, the
analog-to-digital converter generating digital data responsive to
the amplified signal; and
a computer coupled to the analog-to-digital converter, the computer
categorizing the particles responsive to the digital data.
12. The data acquisition system of claim 11 further comprising a
preamplifier coupled between the first detector and the overlap
compensation circuit, the preamplifier receiving the first analog
current from the first detector, the preamplifier generating a
voltage-encoded signal responsive to said first analog current, the
preamplifier generating an estimated DC component of the
voltage-encoded signal, the preamplifier attenuating the
voltage-encoded signal based on a voltage offset and the estimated
DC component to produce a DC compensated signal, the overlap
compensation circuit generating the overlap compensated signal by
subtracting the second signal from the DC compensated signal.
13. The data acquisition system of claim 12 wherein the
preamplifier includes:
a current-to-voltage converter coupled to the first detector, the
current-to-voltage converter receiving the first analog current and
generating the voltage-encoded signal responsive to the analog
current;
a baseline restoration circuit coupled to the current-to-voltage
converter, the baseline restoration circuit estimating the
estimated DC component responsive to the voltage-encoded signal;
and
an offset compensation circuit coupled to the baseline restoration
circuit, the offset compensation circuit generating an offset
signal indicating the voltage offset.
14. The data acquisition system of claim 13 wherein:
the baseline restoration circuit generates a DC component signal
having a voltage level equal to the offset signal summed with the
estimated DC component; and
the DC compensated signal is generated by attenuating the
voltage-encoded signal based on the DC component signal.
15. The data acquisition system of claim 11 wherein said particles
are cells.
16. A data acquisition system for use in a flow cytometry system,
comprising:
a first detector disposed to detect particles having a first tag
type, the first detector generating a first analog current which
varies responsive to detecting particles having the first tag
type;
a first preamplifier coupled to the first detector, the first
preamplifier receiving the first analog current from the first
detector, the first preamplifier estimating a first estimated DC
component of the first analog current based on the first analog
current, the first preamplifier attenuating the first analog
current based on a first voltage offset and the first estimated DC
component to produce a first DC compensated signal;
a first overlap compensation circuit, the first overlap
compensation circuit including
a first reduced signal transmission circuit coupled to the first
preamplifier, the first reduced signal transmission circuit
receiving the first DC compensated signal and generating a first
reduced level signal, the first reduced level signal being the
first DC compensated signal reduced by a first percentage, and
a first subtraction circuit coupled to the first preamplifier, the
first subtraction circuit subtracting a second reduced level signal
from the first DC compensated signal to produce a first overlap
compensated signal;
a first half-wave rectifier coupled to the first overlap
compensation circuit, the first half-wave rectifier receiving the
first overlap compensated signal and generating a first rectified
signal;
a first amplifier coupled to the first half-wave rectifier, the
first amplifier amplifying the first rectified signal to generate a
first amplified signal;
a first analog-to-digital converter coupled to the first amplifier,
the first analog-to-digital converter generating digital data
responsive to the first amplified signal;
a second detector disposed to detect particles having a second tag
type, the second detector generating a second analog current which
varies responsive to detecting particles having the second tag
type;
a second preamplifier coupled to the second detector, the second
preamplifier receiving the second analog current from the second
detector, the second preamplifier estimating a second estimated DC
component of the second analog current based on the second analog
current, the second preamplifier Attenuating the second analog
current based on a second voltage offset and the second estimated
DC component to produce a second DC compensated signal;
a second overlap compensation circuit, the second overlap
compensation circuit including
a second reduced signal transmission circuit coupled to the second
preamplifier and to the first subtraction circuit, the second
reduced signal transmission circuit receiving the second DC
compensated signal and generating the second reduced level signal,
the second reduced level signal being the second DC compensated
signal reduced by a second percentage, and
a second subtraction circuit coupled to the second preamplifier and
to the first reduced signal transmission circuit, the second
subtraction circuit subtracting the first reduced level signal from
the second DC compensated signal to produce a second overlap
compensated signal;
a second half-wave rectifier coupled to the second overlap
compensation circuit, the second half-wave rectifier receiving the
second overlap compensated signal and generating a second rectified
signal;
a second amplifier coupled to the second half-wave rectifier, the
second amplifier amplifying the second rectified signal to generate
a second amplified signal;
a second analog-to-digital converter coupled to the second
amplifier, the second analog-to-digital converter generating
digital data responsive to the second amplified signal;
a computer coupled to the first analog-to-digital converter and to
the second analog-to-digital converter, the computer categorizing
the particles responsive to the digital data generated by the first
analog-to-digital converter and the second analog-to-digital
converter.
17. A method for acquiring data to characterize particles in a flow
cytometry system, the method comprising the steps of:
detecting particles having a first tag type with a first
detector;
generating a first signal responsive to detection of particles
having the first tag type by the first detector;
detecting particles having a second tag type with a second
detector;
generating a second signal responsive to detection of particles
having the second tag type by the second detector;
subtracting a percentage of the second signal from the first signal
to produce an overlap compensated signal;
applying the overlap compensated signal to a half-wave rectifier to
produce a rectified signal;
amplifying the rectified signal to produce an amplified signal;
converting the amplified signal to digital data;
transmitting the digital data to a computer;
causing the computer to characterize particles based on the digital
data.
18. The method of claim 17 farther including the steps of:
estimating an estimated DC component of the first signal;
attenuating the first signal based on a predetermined voltage
offset summed with the estimated DC component prior to subtracting
the percentage of the second signal from the first signal to
produce the overlap compensated signal.
19. A method for performing spectral overlap compensation in a
high-speed flow cytometry system, comprising the steps of:
receiving a first signal from a first detector configured to detect
signals from a first tag type;
receiving a second signal from a second detector configured to
detect signals from a second tag type;
subtracting a percentage of the second signal from the first signal
to produce an overlap-compensated signal; and
rectifying the overlap-compensated signal to eliminate any
undershoot caused during said step of subtracting.
20. The method of claim 19 wherein said step of receiving said
first signal includes:
receiving an analog current from said first detector;
converting said analog current into a voltage-encoded signal;
generating an estimated DC component of said voltage-encoded
signal; and
attenuating the voltage-encoded signal based on a voltage offset
summed with the estimated DC component to produce said first
signal.
21. The method of claim 19 wherein said flow cytometry system has a
pulse rise time of no more than approximately two microseconds.
Description
FIELD OF THE INVENTION
The present invention relates to a flow cytometry system, and more
specifically, to a method and apparatus for compensating for
spectral overlap of fluorochrome emission.
BACKGROUND OF THE INVENTION
Flow cytometry involves serial characterizing of particles, such as
cells or cellular components, in a fluid stream. The particles are
tagged with, for example, a fluorescent dye. The stream of
particles is then passed through a laser beam, causing the tag to
fluoresce. The light pulses from the tag provide an identifying
signature for the particle. The light pulses typically have pulse
widths between three to five microseconds. A detector detects the
pulses from the tag and transmits a signal representing the pulses
to a data acquisition system (DAS). The DAS then categorizes the
particles based on the detected pulses.
Some flow cytometry systems segregate particles based on their
classification after the particles have been classified. This
segregation is performed by causing the stream to break into
droplets. Preferably, each droplet will contain one particle. As
soon as the droplet is formed, the droplet is electrically charged
responsive to the category to which the particle within the droplet
belongs. The droplet then falls between electrostatic plates.
Differently charged droplets will be pulled in different directions
as they travel between the charged plates.
A multiple-laser flow cytometer uses a plurality of spaced beams,
each of a different wavelength, to excite different fluorescent
dyes. Thus, more information can be obtained using a multiple-laser
flow cytometer since each particle can be probed successively by
each beam to provide information relating to a multitude of
characteristics.
Data collection in a multibeam system is complex, because particle
classification involves the cross-correlation of information
detected by two or more different sensors. In cytometer systems
which segregate particles in real time, the data for a particle
must be synchronized and the categorization operation completed
between the time the particle passes a laser and the time the
stream breaks into droplets.
One method for acquiring and synchronizing particle data at high
speed in a multibeam system is disclosed in U.S. Pat. No. 5,150,313
issued to van den Engh et al. on Sep. 22, 1992. According to this
method, analog signals from various detectors are immediately
converted to digital data. The digital data generated by each
detector in response to detecting a given particle is synchronized
with the digital data from the other detectors for the given
particle using FIFO buffers. The synchronized data for the given
particle is then sent sequentially over a digital data bus to a
computer.
As the data acquisition rate of cytometry systems increases, it
becomes increasingly difficult to maintain the integrity of the
data generated by the detectors. In many systems, the electrical
current from the detectors varies in response to detected pulses
from the particles. Therefore, as a first step, the current-encoded
signal generated by each detector is processed by a
current-to-voltage converter to produce a voltage-encoded pulse
signal. Typically, only the peak of the signal is important. The
resulting voltage-encoded pulse signal has an
information-distorting DC component resulting in false values of
peak signal.
Normally, this offset may be eliminated with a baseline restoration
circuit. Baseline restoration circuits are circuits which reduce
the DC voltage of an input signal based on an estimated DC
component. Baseline restoration circuits are typically feedback
circuits. Thus, the estimated DC level used to reduce the voltage
of a voltage-encoded input signal is based on the level of the
current-to-voltage converter's output signal.
FIG. 1 illustrates a typical baseline restoration circuit 100. The
input signal on an input line 102 is reduced based on an estimated
DC component signal on a line 104 to produce a DC-compensated
output signal on a line 106. The estimated DC component signal is
produced by a DC component estimating circuit 108 based on the
DC-compensated output signal on line 106 at junction 110.
The effectiveness of baseline restoration circuit 100 is reduced
when data is acquired a higher frequencies. Specifically, at
frequencies above approximately 5000 events/second and pulse rise
times less than or equal to 2 microseconds, the magnitude of the
DC-component still present in the DC-compensated output signal will
typically be greater than 1 mV. A DC-component of that magnitude is
typically unacceptable due to the sensitivity of the signal
processing circuitry that receives the DC-compensated output
signal. Specifically, the signal processing circuitry that follows
a baseline restoration circuit in a flow cytometry system typically
includes a logarithmic amplifier that is sensitive to input signals
having amplitudes at or greater than 1 mV.
Often, the spectra of two dyes used simultaneously are likely to
have an area or areas of overlap, called spectral overlap. The
process by which the electronics adjusts for spectral overlap is
called "compensation".
Increasing the data acquisition rate in a flow cytometry system may
also result in information distortion during spectral overlap
compensation operations. Spectral overlap compensation is typically
performed to more clearly segregate data distributions acquired in
multiple-detector flow cytometry systems into predefined
quadrants.
Referring to FIG. 2, detection of an X-tagged particle may result
in a spectral response 230, while detection of a Y-tagged particle
may result in a spectral response 232. Some particles will have
both X and Y tags. There is a range of frequencies 234 at which
both X and Y-tagged particles produce output signals. Because of
this overlap, populations may not categorize squarely into
categorization quadrants. However, categorization into quadrants is
preferable for most sorting electronics and/or analysis
software.
Most flow cytometry systems compensate for this overlap by reducing
the signal of one detector by a percentage of a signal from another
detector. This spectral overlap compensation process is described
in the article "Two Color Immunofluorescence Using a
Fluorescence-Activated Cell Sorter", Loken M R, Parks D R,
Herzenberg L A, Journal of Histochem Cytochem, 25:899 (1977).
This spectral overlap compensation method works well for typical
multiple detector flow cytometry systems. However, in systems with
input pulse rise times at or less than 2 .mu.s, it becomes
increasingly difficult to precisely align the two
detector-generated signals to accurately perform the required
signal subtraction operation. Even small signal alignment errors
may cause signal reduction errors, some of which may result in
undershoot portions on the output signal. Such undershoot portions
may significantly distort the information contained in the signal
if they are amplified by a logarithmic amplifier, whose output
signal depends on the absolute value of its input voltage.
Another approach to compensation is described in the paper:
Fluorescence Spectral Overlap Compensation for Any Number of Flow
Cytometry Parameters by C. Bruce Bagwell and Earl G. Adams, Annals
New York Academy of Sciences, pg. 167-184 (1992). This approach is
limited due to the relatively low resolution of analog/digital
conversion in typical flow cytometers, and therefore is useful in a
limited number of applications.
Based on the foregoing, it is clearly desirable to provide a method
and apparatus for use in a high speed data acquisition system for
reducing the DC component. It is further desirable to reduce the DC
component which cannot be completely eliminated due to the
limitations of feedback baseline restoration circuits. In addition,
it would be desirable to reduce the information distortion which
may result from signal alignment errors during spectral overlap
compensation operations.
SUMMARY OF THE INVENTION
A preamplifier for use in a flow cytometry system is provided
according to one aspect of the present invention. The preamplifier
includes a baseline restoration circuit and an offset compensation
circuit. The baseline restoration circuit is coupled to receive a
voltage-encoded signal. The baseline restoration circuit generates
an estimated DC component responsive to the voltage-encoded signal.
The offset compensation circuit is coupled to the baseline
restoration circuit. The offset compensation circuit generates an
offset signal indicating a voltage offset. The preamplifier is
configured to attenuate the voltage-encoded signal based on the
voltage offset and the estimated DC component to produce a DC
compensated signal.
A spectral overlap compensation system for use in a flow cytometry
system is provided according to one aspect of the present
invention. The spectral overlap compensation system includes an
overlap compensation circuit, a half-wave rectifier and an
amplifier. The overlap compensation circuit is coupled to a first
detector. The overlap compensation circuit receives a first signal
from the first detector. The overlap compensation circuit subtracts
a second signal from the first signal to produce an overlap
compensated signal. The second signal is based on signals produced
by one or more other detectors. The half-wave rectifier is coupled
to the overlap compensation circuit. The half-wave rectifier
receives the overlap compensated signal and generates a rectified
signal. The amplifier is coupled to the half-wave rectifier. The
amplifier amplifies the rectified signal and generates an amplified
signal.
The effect of the half-wave rectifier depends on the type of
amplifier employed. For example, a logarithmic amplifier whose
output depends on the absolute value of its input signal requires
the half-wave rectifier to prevent false reading due to signal
undershoots.
According to another aspect of the invention, a data acquisition
system for use in a flow cytometry system is provided. The data
acquisition system includes a detector, a preamplifier, an
amplifier, an analog-to-digital converter and a computer.
The detector is disposed to detect particles having a first tag
type. The detector generates an analog current which corresponds to
detecting particles having the first tag type. The preamplifier is
operatively coupled to the detector. The preamplifier receives the
analog current from the detector. The preamplifier generates a
signal based on the analog current and an estimated DC component of
the signal. In a high-speed data acquisition system, a feedback
baseline restoration circuit will tend to underestimate the DC
component of a signal. Therefore, the preamplifier of the present
invention reduces the DC component based on the sum of the
estimated DC component and a predetermined offset.
The amplifier is operatively coupled to the preamplifier. The
amplifier amplifies the DC compensated signal and generates an
amplified signal. The analog-to-digital converter is operatively
coupled to the amplifier. The analog-to-digital converter generates
digital data responsive to the amplified signal. The computer is
operatively coupled to the analog-to-digital converter. The
computer categorizes the particles responsive to the digital
data.
According to another aspect of the present invention, a data
acquisition system for use in a multiple-detector flow cytometry
system is provided. The data acquisition system includes a first
detector, a second detector, a reduced signal transmission circuit,
an overlap compensation circuit, a half-wave rectifier, an
amplifier, an analog-to-digital converter and a computer.
The first detector is disposed to detect particles having a first
tag type. The first detector generates a first analog current which
corresponds to detecting particles having the first tag type. The
second detector is disposed to detect particles having a second tag
type. The second detector generates a second analog current which
corresponds to detecting particles having the second tag type.
The reduced signal transmission circuit is operatively coupled to
the second detector. The reduced signal transmission circuit
receives the second analog current and generates a reduced level
signal. The reduced level signal represents the second analog
current reduced by a predetermined percentage. The overlap
compensation circuit is operatively coupled to the first detector
and the reduced signal transmission circuit. The overlap
compensation circuit subtracts the reduced level signal from a
first signal representative of the first analog current to produce
an overlap compensated signal.
Due to timing and component imprecision, the overlap compensated
signal produced by this subtraction operation may dip below ground.
If these below-ground dips are not removed, they may result in
erroneous signals after logarithmic amplification. Therefore, the
overlap compensated signal is fed into the half-wave rectifier. The
half-wave rectifier is operatively coupled to the overlap
compensation circuit. The half-wave rectifier receives the overlap
compensated signal and generates a rectified signal in which the
below-ground signal dips have been eliminated.
The log amplifier is operatively coupled to the half-wave
rectifier. The amplifier generates an amplified signal by
amplifying the rectified signal. The analog-to-digital converter is
operatively coupled to the amplifier. The analog-to-digital
converter generates digital data responsive to the amplified
signal. The particles are categorized responsive to the digital
data.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention is illustrated by way of example, and not by
way of limitation, in the figures of the accompanying drawings and
in which like reference numerals refer to similar elements and in
which:
FIG. 1 illustrates a feedback circuit for reducing the DC component
of a signal;
FIG. 2 illustrates the overlap of optical spectra of
differently-tagged particles;
FIG. 3 is a block diagram illustrating the data acquisition portion
of a multiple-detector flow cytometry system according to an
embodiment of the invention;
FIG. 4 is a block diagram illustrating a preamplifier with an
offset compensation circuit according to an embodiment of the
invention;
FIG. 5 is a schematic diagram illustrating the preamplifier of FIG.
4 in greater detail;
FIG. 6 is a schematic diagram illustrating an embodiment of the
overlap compensation circuit and half-wave rectifier of FIG. 3;
FIG. 7 is a flow chart illustrating the steps for acquiring data in
multiple-detector high-speed flow cytometry system; and
FIG. 8 illustrates exemplary signals within the data acquisition
portion of the multiple-detector flow cytometry system illustrated
in FIG. 3.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Referring to FIG. 3, it illustrates a block diagram of a two
parameter data acquisition system 301 for a multiple-detector flow
cytometry system. Typical cytometry systems include five to eight
detectors. However, for the purposes of explanation, data
acquisition system 301 is illustrated with two detectors 302 and
356. Data acquisition system 301 also includes two preamplifiers
300 and 358, two overlap compensation circuits 326 and 364, two
half-wave rectifiers 330 and 374, two logarithmic amplifiers 336
and 378, two analog-to-digital converters 340 and 382, and two
digital first-in-first-out (FIFO) buffers 344 and 386. Data
acquisition system 301 further includes a bus 348, a computer
interface 350, a second bus 352, and a computer 354. Preferably,
detector 302 is configured to detect particles (e.g. cells) with a
first type of tag, and detector 356 is configured to detect
particles with a second type of tag. For the purposes of
explanation, it shall be assumed that detector 302 detects
"X-tagged particles", while detector 356 detects "Y-tagged
particles". Some particles may have both X and Y tags. Such
particles will be detected by both detector 302 and detector
356.
Detector 302 is coupled to preamplifier 300 by a coaxial cable 308.
Detector 302 generates a signal to preamplifier 300 over coaxial
cable 308 indicative of the light detected from the X-tagged
particles. The signal generated by detector 302 is current-encoded.
That is, the current of the signal generated by detector 302 varies
based on the light pulses detected by the detector 302.
Preamplifier 300 includes a current-to-voltage converter for
converting the current-encoded signal to a voltage-encoded pulse
signal, and a feedback baseline restoration circuit for reducing
the DC component of the voltage-encoded pulse signal. However, as
explained above, due to the high data acquisition rate of detector
302, the baseline restoration circuit within preamplifier 300 will
not completely eliminate the DC component on the voltage-encoded
pulse signal. Therefore, preamplifier 300 further includes an
offset compensation circuit 316 to reduce the DC component that
remains on the voltage-encoded pulse signal. The resulting DC
compensated signal is sent over line 324 to overlap compensation
circuit 326.
As described above, there may be an overlap between the optical
spectra of particles with different tags. To provide a more
discrete classification of the tagged particles, overlap
compensation circuit 326 reduces the amplitude of the DC
compensated signal on line 324 by a percentage of a DC compensated
signal on line 370, which encodes information detected by detector
356. A signal representing a fraction of the DC compensated signal
on line 370 is transmitted to overlap compensation circuit 326 by
overlap compensation circuit 364 via a line 366.
Similarly, overlap compensation circuit 364 reduces the DC
compensated signal on line 370 based on a signal generated by
overlap compensation circuit 326 over a line 368. The signal
generated over line 368 by overlap compensation circuit 326 has an
amplitude equal to a fraction of the amplitude of the DC
compensated signal on line 324.
As explained above, the spectral overlap compensation operation
performed by overlap compensation circuit 326 may result in an
output signal on line 328 which, at times, undershoots below
zero-volts. For example, assume that during a particular time
period line 324 carries the signal 802 shown in FIG. 8, and line
370 carries the signal 804 shown in FIG. 8. To compensate for
spectral overlap, overlap compensation circuit 326 subtracts a
fraction (e.g. 30%) of signal 804 from signal 802. Due to circuit
imprecision, the resulting signal in high speed systems may have
negative undershoots, as shown in signal 806. These negative
undershoots are caused by timing and component imprecision in a
high-speed system. If not eliminated, these negative undershoots
will distort the actual measurements and result in positive,
amplified spikes 812 when the overlap-compensated signal is log
amplified through logarithmic amplifier 336, which has an output
that depends on the absolute value of its input signal.
These undershoots are reduced by passing the overlap-compensated
signal through half-wave rectifier 330 before sending the signal to
logarithmic amplifier 336. Half-wave rectifier 330 receives the
signal 806 over line 328 and eliminates undershoot portions of the
signal. The resulting rectified signal is then passed to
logarithmic amplifier 336 over line 332 to produce an amplified
signal 810.
Logarithmic amplifier 336 amplifies the rectified signal and sends
the resulting amplified signal to analog-to-digital converter 340
over line 338. Analog-to-digital converter 340 converts the
amplified signal on line 338 to a digital signal and transmits the
digital signals over line 342 to FIFO buffer 344. FIFO buffer 344
receives the digital signals on line 342 and transmits the digital
data contained in the signal to computer 354 over buses 348 and 352
through computer interface 350. FIFO buffer 344 aligns the digital
data contained therein with the digital data stored in FIFO buffer
386 so that data corresponding to the same particle but detected by
different detectors is sent to bus 348 sequentially by FIFO buffer
344 and FIFO buffer 386.
In a particle-sorting flow cytometry system, each particle is
categorized based on the data received from data acquisition system
301. Each particle is then electrically charged and segregated
based on the category of the particle.
Detector 356 is coupled to preamplifier 358 by a line 357.
Preamplifier 358 includes an offset compensation circuit 360.
Overlap compensation circuit 364 is coupled to preamplifier 358 by
a line 370, to overlap compensation circuit 326 by line 366 and
368, and to half-wave rectifier 374 by a line 372. Logarithmic
amplifier 378 is coupled to half-wave rectifier by a line 376, to
analog-to-digital converter 382 by a line 380. FIFO buffer 386 is
coupled to analog-to-digital converter 382 by a line 384, and to
bus 348 by a line 380.
Detector 356 and its associated circuitry work as described above
with respect to detector 302 and its associated circuitry. In
particular, offset compensation circuit 360 reduces the DC
component which is not otherwise eliminated by a baseline
restoration circuit in preamplifier 358. Also, similar to half-wave
rectifier 330, half-wave rectifier 374 eliminates any voltage
undershoots on the voltage-encoded pulse signal generated by
overlap compensation circuit 364 before transmitting the overlap
compensated signal to logarithmic amplifier 378.
Data acquisition system 301 overcomes high-speed related signal
distortion by use of offset compensation circuits 316 and 360, as
well as half-wave rectifiers 330 and 374. Specifically, offset
compensation circuits 316 and 360 reduce the DC component not
eliminated by baseline-restoration circuits, and half-wave
rectifiers 330 and 374 eliminate signal undershoots caused by
signal alignment errors in overlap compensation operations. Because
data acquisition system 301 includes these additional circuits, the
integrity of the data ultimately received by computer 354 or
sorting circuitry is significantly increased.
Referring now to FIG. 4, it illustrates preamplifier 300 in greater
detail. Preamplifier 300 generally includes a current-to-voltage
converter 404, a baseline restoration circuit 406, a signal buffer
422, and offset compensation circuit 316. Current-to-voltage
converter 404 generates a voltage-encoded pulse signal over a line
410. The voltage level of voltage-encoded pulse signal varies
responsive to the current of the current-encoded signal on coaxial
cable 308. The voltage-encoded pulse signal is transmitted to
baseline restoration circuit 406 over line 410.
Baseline restoration circuit 406 estimates the DC component of the
signal on line 410. An estimated DC component signal is transmitted
back to current-to-voltage converter 404 over a line 412.
Current-to-voltage converter 404 then reduces the voltage-level
encoded signal by the estimated DC component amount and transmits
the compensated signal to signal buffer 422 over a line 414 for
further processing.
As explained above, at high speed there is an inaccuracy between
the signal on line 410 used to estimate the DC component, and the
signal which is reduced by the DC component estimate. Consequently,
the DC component would typically not be adequately reduced from the
output signal on line 414. However, adjustable offset compensation
circuit 316 is coupled to baseline restoration circuit 406 by a
line 420. Adjustable offset compensation circuit 316 causes
baseline restoration circuit 406 to alter (usually increase) the
amplitude of the DC component estimate signal sent over line 412 by
a predetermined amount. In the preferred embodiment, adjustable
offset compensation circuit 316 increases the estimated DC
component estimate signal on line 412 by an mount sufficient to
more accurately reduce the DC component from the signal ultimately
generated at line 414. From signal buffer 422, the DC compensated
signal is then sent to overlap compensation circuit 326 as
described above.
Referring to FIG. 5, it is a schematic diagram illustrating one
embodiment of preamplifier 300. In the illustrated embodiment,
current-to-voltage converter 404 includes a operational amplifier
510 with an output 522 and two inputs 508 and 512. Input 512 of
operational amplifier 510 is coupled to coaxial cable 308 through a
input protection resistor 502. Input 512 of operational amplifier
510 is coupled to ground through protection diode 504, to ground
through diode 506, to the output 522 of operational amplifier 510
through a resistor 516, to the output 522 through a bandwidth
limiting capacitor 518, and to line 412 through a resistor 520.
Input 508 of operational amplifier 510 is coupled to ground.
Operational amplifier 510 is also coupled to ground through a
compensation capacitor 514. Through this arrangement of components,
current-to-voltage converter 404 generates a signal on line 414
that has a voltage which varies in response to the current on
coaxial cable 308. The voltage level on line 414 has been reduced
responsive to the DC component estimate signal on line 412 and by
offset compensation circuit 316 to reduce the DC component from the
signal.
Baseline restoration circuit 406 generates the DC-offset estimate
signal on line 412 in response to the voltage encoded signal on
line 410 and a signal from adjustable offset compensation circuit
316. Baseline restoration circuit 406 includes a operational
amplifier 524 with two inputs 526 and 528 and an output 530.
Baseline restoration circuit 406 also includes a differential
integrator 531 with two inputs 532 and 534 and an output 536. Input
526 of operational amplifier 524 is coupled to the output 530 of
operational amplifier 524 through a resistor 538, through a diode
540, and through a diode 542. Input 526 of operational amplifier
524 is also coupled to line 410 through a resistor 544. Input 528
of operational amplifier 524 is coupled to ground. Output 530 of
operational amplifier 524 is coupled to input 532 of differential
integrator 531 through a resistor 546. Input 532 of differential
integrator 531 is also coupled to output 536 of differential
integrator 531 through a capacitor 548. Configured as described
above, these components of baseline restoration circuit generate a
signal over line 412 that has a voltage level equal to the
estimated DC component of the voltage encoded signal on line 410.
However, due to the limitations at higher pulse input rates between
the signal adjusted based upon the signal on line 412 and the
signal on line 410 from which the signal on line 412 is generated,
baseline restoration circuit 406 must be adjusted by adjustable
offset compensation circuit 316 to more accurately reduce the DC
component from the signal generated on line 414.
Adjustable offset compensation circuit 316 is connected to "offset
null" provision of operational amplifier 524 in baseline
restoration circuit 406 by a plurality of lines 552 and 554.
Adjustable offset compensation circuit 316 includes a potentiometer
556 which includes a resistor 560. The signal sent from
potentiometer 556 to baseline restoration circuit 406 causes the
voltage level of the DC-offset estimate signal on line 412 to be
altered by an amount based on the setting of potentiometer 556.
Preferably, potentiometer 556 is set to a level that will alter the
DC-offset estimate signal on line 412 enough to more accurately
reduce the DC component from the signal generated over line
324.
Signal buffer 422 buffers the signal on line 414 and drives the
signal to overlap compensation circuit 326 via line 324. Signal
buffer 422 includes a operational amplifier 562 with two inputs 564
and 566 and output 570. Input 564 is coupled to output 570. Input
566 is coupled to line 414 through a resistor 568. Output 570 is
coupled to line 324 through a resistor 572.
In certain situations, it may be desired to know the DC component
portion of signals generated by detectors in a flow cytometry
system. As explained above, an estimate of this component is
generated by baseline restoration circuit 406 over line 412.
Optionally, baseline restoration circuit 406 may send the DC-offset
estimate signal over a line 550 for processing by other circuits in
addition to current-to-voltage converter 440.
Referring to FIG. 6, it is a schematic diagram illustrating overlap
compensation circuit 326 and half-wave rectifier 330 of FIG. 3 in
greater detail. Overlap compensation circuit 326 includes a buffer
602, a reduced signal transmission circuit 614, a switch 620, and a
signal reduction circuit 621. Buffer 602 includes an operational
amplifier 604 with two inputs 606 and 608 and an output 611. Input
608 of operational amplifier 604 is connected to line 324, and
input 606 of operational amplifier 604 is connected to output 611
of operational amplifier 604. A potentiometer and a resistor 612
can be connected to a operational amplifier 604. The output 611 of
operational amplifier 604 is coupled to signal subtraction circuit
621 by a line 622. Thus configured, buffer 602 buffers and drives
the signal transmitted to overlap compensation circuit 326 by
preamplifier 300 over line 324. The same result can be achieved by
offset adjustment in the preamplifier.
Reduced signal transmission circuit 614 is connected to line 622.
Reduced signal transmission circuit 614 is configured such that a
percentage of the voltage on line 622 is transmitted to overlap
compensation circuit 364 via line 368. The voltage on line 622
varies in response to the signal generated by detector 302.
Detector 302 is configured to detect X-tagged particles. Detector
356 is configured to detect particles which are tagged differently
than those detected by detector 302, which detects Y-tagged
particles. However, even though detector 302 is configured to
detect particles with a different type of tag, detector 302 may
generate false detection indications because the light frequencies
generated by the particles detectable by detector 356 partially
overlap the frequencies emitted by the particles detectable by
detector 302. As a result, the voltage on line 622 may increase
when a Y-tagged particle passes by detector 302. To eliminate these
false detection readings, signal reduction circuit 621 reduces the
signal on line 622 with the signal on line 366. Overlap
compensation circuit 364 subtracts the reduced signal on line 368
from a DC offset compensated signal from detector 356.
Line 366 carries a signal whose voltage is a percentage of the
voltage-encoded pulse signal generated by preamplifier 358. Signal
reduction circuit 621 includes a operational amplifier 630 with two
inputs 631 and 633 and an output 618. Input 633 of operational
amplifier 630 is connected to switch 620 through a resistor 624,
and to ground through a resistor 626. Input 633 is coupled to line
366 through switch 620 when switch 620 is on, and is coupled to
ground through switch 620 when switch 620 is off. Input 631 of
operational amplifier 630 is coupled to line 622 through a resistor
628, and the output 618 of operational amplifier 630 through a
resistor 632. An offset adjustment circuit 634, which includes a
resistor 636, is also coupled to operational amplifier 630. When
switch 620 is on, signal reduction circuit 621 generates a signal
over line 328 based on the signal on line 622 subtracted by the
signal on line 366.
Half-wave rectifier 330 generally includes a operational amplifier
642 with two inputs 644 and 646 and an output 643. Input 644 of
operational amplifier 642 is coupled to line 328 through resistor
640, to line 322 through a resistor 652, and to the output 643 of
operational amplifier 642 through a diode 650. Input 646 of
operational amplifier 642 is coupled to ground. The output of
operational amplifier 642 is coupled to line 332 through a diode
654. Operational amplifier 642 is also coupled to a potentiometer
648.
Half-wave rectifier 330 receives the signal on line 328 from
overlap compensation circuit 326. The signal on line 328 coming
from overlap compensation circuit 326 has a negative polarity.
Half-wave rectifier 330 eliminates any above-zero voltage
undershoots in the signal and transmits the resulting rectified
signal over line 332. The rectified signal generated by half-wave
rectifier 330 has a positive polarity.
As explained above, the signal reduction operation performed by
signal reduction circuit 621 may result in signal spikes on line
328 above ground due to offset and analog component imprecision.
Eliminating such above-zero signal undershoots by half-wave
rectifier 330 will ensure that false signal spikes will not be
present after the signal is log amplified by logarithmic amplifier
336.
The embodiments of preamplifier 300, overlap compensation circuit
326, and half-wave rectifier 330 described above with reference to
FIGS. 5 and 6 may implemented with components having the
operational parameters shown in Table 1. It should be understood
that the values shown in Table 1, as well as the specific circuitry
configurations described above, are merely exemplary. The invention
may be implemented by a variety of circuitry configurations.
TABLE 1 ______________________________________ resistor 568 100
.OMEGA. input protection resistor 502 200 .OMEGA. protection diode
504 Item 1N4148 available from Motorola diode 506 Item 1N4148
available from Motorola operational amplifier 510 Item AD829JN
available from Analog Devices compensation capacitor 514 4 pF
resistor 516 20K capacitor 518 4 pF resistor 520 20K operational
amplifier 524 Item AD744JN available from Analog Devices
differential integrator 531 Item AD744JN available from Analog
Devices resistor 538 100K diode 540 Item 1N4148 available from
Motorola resistor 544 1K resistor 546 24.9K capacitor 548 0.1 .mu.F
potentiometer 556 2M resistor 560 1M operational amplifier 562 Item
AD847JN available from Analog Devices resistor 572 100 .OMEGA.
operational amplifier 604 Item AD845JN available from Analog
Devices resistor 612 392 .OMEGA. + 1K potentiometer potentiometer
614 10K resistor 624 10K resistor 626 10K resistor 628 10K
operational amplifier 630 Item AD845JN available from Analog
Devices resistor 632 10K potentiometer 634 1K resistor 636 392
.OMEGA. + 1K potentiometer resistor 640 1K operational amplifier
642 Item AD844AN available from Analog Devices potentiometer 648
20K diode 650 Item HP5082-2810 available from Hewlett Packard
resistor 652 1K diode 654 Item HP5082-2810 available from Hewlett
Packard ______________________________________
Referring now to FIG. 7, it is a flow chart illustrating a method
for acquiring data to characterize particles in a flow cytometry
system according to one embodiment of the invention. At step 700A,
particles having a first tag type (e.g. X-tagged particles) are
detected with a first detector. At step 702A, a first signal is
generated responsive to detection of X-tagged particles. In the
preferred embodiment, the first signal is a current-encoded
signal.
At step 708A, the DC component of the first signal is estimated
based on the first signal. Because the method is for use with
high-speed data acquisition, a feedback circuit will typically
underestimate the DC component. Therefore, at step 710A, the first
signal is DC compensated based on a voltage offset summed with the
estimated DC component. The voltage offset is adjustable.
A second signal is generated and processed concurrent with the
generation and processing of the first signal. The second signal is
a signal from a second detector that detects particles having a
second tag type (e.g. Y-tagged particles). Specifically, at steps
700B and 702B, a second signal is generated responsive to detection
Y-tagged particles by the second detector, and at steps 708B and
710B the second signal is DC compensated based on a voltage offset
summed with an estimated DC component.
At steps 712A and 712B, two overlap compensated signals are
generated based on the first signal and the second signal.
Specifically, at step 712A, a percentage of the second signal is
subtracted from the first signal to produce a first overlap
compensated signal. At step 712B, a percentage of the first signal
is subtracted from the second signal to produce a second overlap
compensated signal.
At steps 714A and 714B, the overlap compensated signals are applied
to half-wave rectifiers. At steps 716A and 716B, the rectified
signals are log amplified to produce amplified signals. At steps
718A and 718B, the amplified signals are converted to digital data.
At step 720, the digital data is transmitted to a computer.
Finally, at step 722, the particles are characterized based on the
digital data.
In the multiple-detector system shown in FIG. 3, the circuits which
process the signal from detector 356 include preamplifier 358 with
offset compensation circuit 360, overlap compensation circuit 364
which compensates the signal from detector 356 by a percentage of
the signal from detector 302, and half-wave rectifier 374 which
eliminates any signal undershoots caused by the overlap
compensation circuit due to timing and component imprecision.
Once stored in FIFO buffers 344 and 386, the digital data from
detectors 302 and 356 are aligned so that the data from each
detector for a given particle is sequentially placed on bus 348.
Each particle may be characterized based on the signal generated by
detectors 302 and 356 responsive to the signal. In a
particle-sorting system, a sorting mechanism is activated to
segregate the particles based on the determined
characterization.
ELECTRONIC BENCH TESTS
The circuitry described above may be adjusted as follows. First,
tie line 308 to ground. Connect a digital volt meter (DVM) with a
minimum of 0.1 mV resolution to the output 611 of operational
amplifier 604. Adjust offset compensation circuit 316 so that the
DVM reads 0.0 V. Apply a Gaussian pulse shape of 2 microseconds
length, 10 kHz frequency to lines 308 and 357. Adjust the amplitude
of the pulse shape to measure 2 mV top-top at line 622 of overlap
compensation circuit 326 and at the corresponding line of overlap
compensation circuit 364. Set compensation adjustment to 100% by
adjusting potentiometer 614 in overlap compensation circuit 326 and
the corresponding potentiometer in overlap compensation circuit
364. Set switch 620 and the corresponding switch in overlap
compensation circuit 364 to the "compensation on" position. Adjust
potentiometer 634 so that output 328 measures 0.0 mV. Adjust the
corresponding potentiometer in overlap compensation circuit 362 so
that output 376 measures 0.0 mV.
The half-wave rectifier 330 may be adjusted using the same input
signals to lines 308 and 357 as described above. Overlap
compensation circuit 364 is adjusted so that the signal on line 366
is at 50%. View signal 332 on an oscilloscope. A positive 1 mV
top-top pulse should be measured. Adjust potentiometer 648 when an
offset is present so that 1 mV top--top pulse is measured with no
offset. Use a similar method to adjust half-wave rectifier 374.
Potentiometer 614 and the corresponding potentiometer in overlap
compensation circuit 364 are set using calibration beads or
samples.
While specific embodiments of the present invention have been
described, various modifications and substitutions will become
apparent by this disclosure. For example, DC offset compensation
has been described with respect to two fluorescence channels.
However, the in invention is not limited to any particular number
of channels and may readily be expanded to handle compensation
between any number of channels. Such modifications and
substitutions are within the scope of the present invention, and
are intended to be covered by the following claims.
* * * * *